Mammalian fetal lung development is a highly coordinated process of growth and differentiation of airways, parenchymal tissue, and vessels. The developing airway epithelial cells actively secrete chloride ions, which drive the accumulation of fluid and the development of a critical distending airway pressure that promotes differentiation. During the initial 2-year period of this 3-year R01 award, the laboratory focused on the ClC-2 pH- and voltage-activated chloride channel, which we showed was highly expressed in fetal rat lung airway epithelia and rapidly down regulated at birth. We next showed that ClC-2 protein expression in fetal and adult rat lung type II epithelial lines is exquisitely sensitive to SP1 and SP3 expression. The goal of this competitive renewal is to determine the consequences of regulated over-expression or elimination of ClC-2 to lung development and to the pathogenesis of diseases caused by chloride channel malfunction such as cystic fibrosis. The hypothesis is that chloride channel species redundancy in fetal mouse airways protects against malfunction of a single type of chloride channel.
Aim 1 is to examine endogenous regulation of ClC-2 expression in adult and fetal airway epithelial cell lines. The hypothesis in Aim 1 is that phosphorylation and dephosphorylation of SP-1 and SP-3 transcription factors is a major factor controlling the level of ClC-2 mRNA and protein expression. Furthermore, it is hypothesized that control of ClC-2 gene and protein expression in turn regulates chloride transport phenotype.
Aim 2 is to complete characterization of the doxycycline-regulated epithelial-specific hClC-2 mouse model that has been generated in our laboratory during the previous 2-year period. This new mouse model will be critical to defining the safe window for ClC-2 over-expression, and define the maximum level of chloride transport function deliverable to the mouse nasal airway. Mice carrying a k18-rtTA, a tetON-hClC2, or both were generated in the laboratory for these experiments.
Aim 3 is to manipulate ClC-2 expression in CF mice to modulate disease phenotype. We will test the hypothesis that knocking two chloride channels out will lead to immature lungs at birth or murine lung inflammation at baseline or both. Combining the hClC-2 TET-On model with the CF knockout will be the approach for the second hypothesis that CF might be treated by over-expression of hClC-2.
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